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Quantitative Cu-Homeostasis in Live Mammalian Cells at the Single-Molecule Level

单分子水平上活哺乳动物细胞的定量铜稳态

基本信息

批准号：
10618850
负责人：
Tai-Yen Chen
金额：
$ 38.06万
依托单位：
UNIVERSITY OF HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-15 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10618850
关键词：
Achievement Affect Bacteria Behavior Biochemical Biological Assay Biological Process Cells Collaborations Complex Copper DNA-Protein Interaction Disease Environment Equilibrium Family Fluorescence Foundations Genetic Transcription Goals Hepatocyte Hepatolenticular Degeneration Homeostasis Human Imaging Techniques In Vitro Individual Knowledge Mammalian Cell Measurement Mediating Membrane Metalloproteins Metals Mission Mutation National Institute of General Medical Sciences Pathogenesis Pathway interactions Prevention Proteins Public Health Research Roentgen Rays Role Spatial Distribution Synchrotrons Technology Therapeutic Wilson disease protein efflux pump experimental study fluorescence imaging imaging approach induced pluripotent stem cell innovation insight knowledge base live cell imaging novel programs reconstitution response secretion process single molecule single-molecule FRET spatiotemporal stem cells superresolution microscopy ultra high resolution uptake

项目摘要

PROJECT SUMMARY/ABSTRACT Understanding responsive mechanisms of metalloproteins is key to elucidate biological functions of copper (Cu) and to identify the causes of diseases resulting from abnormal metal homeostasis. The cellular Cu uptake and secretion require relevant metalloproteins to adjust in a spatiotemporally coordinated manner to assure proper cellular Cu level. However, in the Cu field, little is known about how metalloproteins are individually regulated nor systematically cooperate with each other in their native environment, i.e., in cells. Our research goal is to understand the responsive mechanisms of Cu-uptake and secretory metalloproteins in live mammalian cells, with specific focuses on how metalloproteins adjust their behaviors such as spatial distributions, oligomeric states, inter-protein and inter-domain interactions for proper Cu balance in a spatiotemporally defined manner. Previous achievements of the PI include discoveries of novel mechanisms of MerR-family metalloregulators in regulating transcription and Cu-responsive dynamic assembly of efflux pumps by examining the protein-DNA interaction and protein diffusive behaviors in live bacteria using single-molecule super-resolution microscopy. Leveraging the power of these technologies, in combined with the recently developed live-cell single-molecule fluorescence- resonance-energy-transfer assay, we will elucidate the responsive mechanisms of metalloproteins in the uptake and secretory pathways in live mammalian cells. Using CTR1 and ATOX1-ATP7A/B as the initial examples of uptake and secretory metalloproteins, the proposed experiments will (1) quantify Cu-dependent oligomeric state distribution and identify the Cu-responsive moiety of CTR1; (2) define the preferential interaction of ATOX1 to ATP7A and ATP7B and how mutations in ATP7B affect Cu homeostasis in cellular Cu defending using induced pluripotent stem cells derived hepatocytes. In addition to primary approaches of single-molecule super-resolution fluorescence imaging techniques, complementary bulk spectroscopic and biochemical measurements will be compared. The research program is further enhanced by collaborations with the experts in Cu homeostasis and stem cell fields. The research is significant because it will provide mechanistic insights into metalloprotein- mediated Cu-uptake and secretion processes as well as complementary information for synchrotron X-ray fluorescence studies on intracellular Cu-redistribution. The comparison between human induced pluripotent stem cell (hiPSC)-derived healthy and diseased hepatocytes will inform how disease mutations disrupt cellular Cu balance, providing the knowledge base to devise therapeutic strategies for Wilson's diseases. The research is innovative because it represents a substantive departure from the status quo by shifting focus to define response mechanisms of metalloproteins using advanced approaches including single-molecule super-resolution microscopy and hiPSC-derived hepatocytes. The live-cell imaging approach also circumvents the general challenge in studying membrane complexes, whose in vitro reconstitution is technically demanding. The hiPSC- derived diseased hepatocytes provide an ideal platform to study the pathogenesis of Wilson's disease.

项目概要/摘要了解金属蛋白的响应机制是阐明铜 (Cu) 生物学功能的关键并确定金属稳态异常导致疾病的原因。细胞对铜的吸收和分泌需要相关金属蛋白以时空协调的方式进行调整，以确保适当的分泌细胞铜水平。然而，在铜领域，人们对金属蛋白如何单独调控知之甚少。也不能在其原生环境（即细胞中）中系统地相互合作。我们的研究目标是了解活哺乳动物细胞中铜吸收和分泌金属蛋白的反应机制，特别关注金属蛋白如何调整其行为，例如空间分布、寡聚状态、蛋白质间和结构域间相互作用，以时空定义的方式实现适当的铜平衡。以前的 PI 的成就包括发现 MerR 家族金属调节剂在调节中的新机制通过检查蛋白质-DNA 相互作用，进行外排泵的转录和 Cu 响应动态组装使用单分子超分辨率显微镜观察活细菌中的蛋白质扩散行为。杠杆作用这些技术的力量，结合最近开发的活细胞单分子荧光- 共振能量转移测定，我们将阐明金属蛋白在摄取中的响应机制和活哺乳动物细胞中的分泌途径。使用 CTR1 和 ATOX1-ATP7A/B 作为初始示例摄取和分泌金属蛋白，拟议的实验将（1）量化铜依赖性寡聚状态分布并鉴定 CTR1 的 Cu 响应部分； (2)定义ATOX1的优先相互作用 ATP7A 和 ATP7B 以及 ATP7B 突变如何影响细胞铜防御中的铜稳态多能干细胞源自肝细胞。除了单分子超分辨率的主要方法之外荧光成像技术、互补的批量光谱和生化测量将比较的。通过与铜稳态专家的合作，该研究计划得到进一步加强。干细胞领域。这项研究意义重大，因为它将提供对金属蛋白的机制见解介导的铜吸收和分泌过程以及同步加速器 X 射线的补充信息细胞内铜重新分布的荧光研究。人诱导多能干细胞的比较细胞 (hiPSC) 衍生的健康和患病肝细胞将告知疾病突变如何破坏细胞铜平衡，为制定威尔逊氏病的治疗策略提供知识基础。该研究是创新，因为它通过将重点转移到定义响应来代表对现状的实质性偏离使用先进方法（包括单分子超分辨率）研究金属蛋白的机制显微镜检查和 hiPSC 衍生的肝细胞。活细胞成像方法还规避了一般的研究膜复合物的挑战，其体外重构在技术上要求很高。 hiPSC- 衍生的患病肝细胞为研究威尔逊病的发病机制提供了理想的平台。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Probing Oxidant Effects on Superoxide Dismutase 1 Oligomeric States in Live Cells Using Single-Molecule Fluorescence Anisotropy.

DOI：
10.1021/cbmi.3c00002
发表时间：
2023-04-24
期刊：
Chemical & biomedical imaging
影响因子：
0
作者：
Chen, Huanhuan;Chen, Tai-Yen
通讯作者：
Chen, Tai-Yen

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system.

DOI：
10.1098/rsob.210128
发表时间：
2021-12
期刊：
Open biology
影响因子：
5.8
作者：
Wen MH;Xie X;Huang PS;Yang K;Chen TY
通讯作者：
Chen TY

Dissection of Interaction Kinetics through Single-Molecule Interaction Simulation.

DOI：
10.1021/acs.analchem.0c01014
发表时间：
2020-09-01
期刊：
Analytical chemistry
影响因子：
7.4
作者：
Pan M;Zhang Y;Yan G;Chen TY
通讯作者：
Chen TY

Generation of a homozygous knock-in human embryonic stem cell line expressing mEos4b-tagged CTR1.

DOI：
10.1016/j.scr.2022.102845
发表时间：
2022-08
期刊：
STEM CELL RESEARCH
影响因子：
1.2
作者：
Chen, Yi-Hung;Huang, Pei-San;Wen, Meng-Hsuan;Pan, Manhua;Lee, Dung-Fang;Chen, Tai-Yen
通讯作者：
Chen, Tai-Yen

Single-molecule microscopy for in-cell quantification of protein oligomeric stoichiometry.